Multidither coherent optical adaptive technique for deep tissue two-photon microscopy

Abstract

Two-photon microscopy normally suffers from the scattering of the tissue in biological imaging. Multidither coherent optical adaptive technique (COAT) can correct the scattered wavefront in parallel. However, the determination of the corrective phases may not be completely accurate using conventional method, which undermines the performance of this technique. In this paper, we theoretically demonstrate a method that can obtain more accurate corrective phases by determining the phase values from the square root of the fluorescence signal. A numerical simulation model is established to study the performance of adaptive optics in two-photon microscopy by combining scalar diffraction theory with vector diffraction theory. The results show that the distortion of the wavefront can be corrected more thoroughly with our method in two-photon imaging. In our simulation, with the scattering from a 450-[Formula: see text]m-thick mouse brain tissue, excitation focal spots with higher peak-to-background ratio (PBR) and images with higher contrast can be obtained. Hence, further enhancement of the multidither COAT correction performance in two-photon imaging can be expected.

FDISCO+: a clearing method for robust fluorescence preservation of cleared samples

“…[6][7][8] However, due to the cloudy features of biological tissue, imaging depth is limited among numerous optical fluorescence microscopes such as the confocal microscope, multi-photon microscope, and light-sheet microscope. 1,[9][10][11][12] In recent years, this problem has been partially solved thanks to the rapid development of optical clearing methods, which can reduce the light scattering and light absorption in the tissues to achieve deep imaging in combination with optical fluorescence microscopy. 13,14 Over the years, a variety of optical clearing methods have been developed.…”